The stem cells (SCs) of the corneal epithelium located in the limbal basal layer are the ultimate source of maintaining corneal epithelial homeostasis. Clinically, loss of limbal SCs or dysfunction of the limbal niche leads to corneal blindness due to limbal SC deficiency (LSCD). During the last funding period, our studies of three surrogate niches, i.e., a serum-free, matrix- free, and fibroblast-free niche, amniotic membrane (AM) niche, and limbal explant niche, have identified several major pitfalls in six protocols currently practiced in treating human patients with LSCD. To circumvent these pitfalls, we have discovered a clever method of isolating the entire limbal epithelial SCs together with their native niche cells (NCs) using collagenase alone. This new isolation method allows us to develop two in vitro model systems for gaining mechanistic understandings of how limbal epithelial SCs are regulated by their native NCs. The first model system breaks SC-NC contact but facilitates their reunion in vitro to generate sphere growth. Using this model system, we will examine whether SC quiescence starts with the SC- NC reunion via binding between stromal derived factor 1 SDF-1 ) expressed by NCs and its receptor (CXCR4) expressed by SCs. Moreover, we will prove that such SC-NC reunion is facilitated by bone morphogenetic protein (BMP) signaling that upregulates expression of SDF- 1 in NCs, and subsequently promotes SC renewal, while further SC renewal can be promoted by Wnt/ -catenin signaling and the FGF family signaling, but suppressed by TGF- signaling (Aim 1). We will also determine whether maintenance of the normal NC phenotype with a small round shape and expression of embryonic SC markers depends on not only biochemical but also physical properties of the basement membrane. By manipulating the basement membrane matrix component and integrity, we expect to isolate NCs and unravel a new therapeutic paradigm for treating LSCD by restoring the limbal niche health through maintenance of the normal NC phenotype (Aim 2). The introduction of exogenous mouse or human labeled cells at the time of SC-NC reunion also allows us to trace the lineage during sphere growth to understand how a normal corneal fate decision is controlled by NCs (Aim 3). Under the limbal niche influence, these studies will resolve a recent controversy whether SCs might reside in the mouse central cornea. Furthermore, they will help resolve whether conjunctival transdifferentiation can take place at the single cell level, and whether oral mucosal and epidermal epithelial progenitors have any potential to take a corneal fate. Taking advantage of an expanding repertoire of transgenic mice, we may turn this model system into a powerful assay to ascertain how human relevance can be drawn from gain or loss of a given gene function during the corneal fate decision. Finally, the aforementioned new knowledge can be applied to the second model system, which maintains SC-NC close contact during isolation and expansion by culturing collagenase-isolated limbal clusters directly on epithelially-denuded AM (Aim 4). This study will lead us achieve the ultimate goal of developing a new tissue engineering strategy to maximize ex vivo expansion of SCs by successful recapitulation of the regulatory mechanism executed by the in vivo native niche. Such an approach may one day help realize the considerable promise held by adult SCs in treating a number of diseases in the body.